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Background: The incidence of hospital-acquired infections in extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) has been increasing worldwide and is frequently associated with an increase in mortality and morbidity rates. The aim of this study was to characterize clinical XDR-PA isolates recovered during six months at three different hospitals in Egypt., Results: Seventy hospital-acquired clinical isolates of P. aeruginosa were classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR), according to their antimicrobial resistance profile. In addition, the possession of genes associated with mobile genetic elements and genes encoding antimicrobial resistance determinants among isolates were detected using polymerase chain reaction. As a result, a significant percentage of the isolates (75.7%) were XDR, while 18.5% were MDR, however only 5.7% of the isolates were non-MDR. The phenotypic detection of carbapenemases, extended-spectrum β-lactamases (ESBLs) and metallo β-lactamase (MBL) enzymes showed that 73.6% of XDR-PA isolates were carbapenemases producers, whereas 75.5% and 88.7% of XDR-PA isolates produced ESBLs and MBL respectively. In addition, PCR screening showed that oxa gene was the most frequently detected gene of carbapenemases (91.4%), while aac(6')-lb gene was mostly detected (84.3%) among the screened aminoglycosides-resistance genes. Furthermore, the molecular detection of the colistin resistance gene showed that 12.9% of isolates harbored mcr-1 gene. Concerning mobile genetic element markers (intI, traA, tnp513, and merA), intI was the highest detected gene as it was amplified in 67 isolates (95.7%). Finally, phylogenetic and molecular typing of the isolates via ERIC-PCR analysis revealed 10 different ERIC fingerprints., Conclusion: The present study revealed a high prevalence of XDR-PA in hospital settings which were resistant to a variety of antibiotics due to several mechanisms. In addition, 98% of the XDR-PA clinical isolates contained at least one gene associated with movable genetic elements, which could have aided the evolution of these XDR-PA strains. To reduce spread of drug resistance, judicious use of antimicrobial agents and strict infection control measures are therefore essential., (© 2024. The Author(s).)

Background: Proteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms., Methods: Antimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated., Results: Antimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where bla TEM , bla SHV , bla CTX-M , bla CIT-M and bla AmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where bla OXA-48 and bla NDM-1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-bla OXA-10 -aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2)., Conclusion: P. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains., (© 2024. The Author(s).)

The spread of fluoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical health threat. This study aims to overcome FQ resistance in A. baumannii via the formulation of polymeric nanoFQs. Herein, 80 A. baumannii isolates were obtained from diverse clinical sources. All A. baumannii isolates showed high resistance to most of the investigated antimicrobials, including ciprofloxacin (CIP) and levofloxacin (LEV) (97.5%). FQ resistance-determining regions of the gyrA and parC genes were the most predominant resistant mechanism, harbored by 69 (86.3%) and 75 (93.8%) of the isolates, respectively. Additionally, plasmid-mediated quinolone resistance genes aac(6')-Ib and qnrS were detected in 61 (76.3%) and 2 (2.5%) of the 80 isolates, respectively. The CIP- and LEV-loaded poly ε-caprolactone (PCL) nanoparticles, F CIP and F LEV , respectively, showed a 1.5-6- and 6-12-fold decrease in the MIC, respectively, against the tested isolates. Interestingly, the time kill assay demonstrated that MICs of F CIP and F LEV completely killed A. baumannii isolates after 5-6 h of treatment. Furthermore, F CIP and F LEV were found to be efficient in overcoming the FQ resistance mediated by the efflux pumps in A. baumannii isolates as revealed by decreasing the MIC four-fold lower than that of free CIP and LEV, respectively. Moreover, F CIP and F LEV at 1/2 and 1/4 MIC significantly decreased biofilm formation by 47-93% and 69-91%, respectively. These findings suggest that polymeric nanoparticles can restore the effectiveness of FQs and represent a paradigm shift in the fight against A. baumannii isolates., (© 2024. The Author(s).)

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) systems are considered as acquired immune mechanisms in Gram-positive and Gram-negative bacteria and also in archaea. They provide resistance/immunity to attacking bacteriophages or mobile genetic elements as integrative conjugative elements (ICE) as well as plasmid transformation. As an opportunistic pathogen, Pseudomonas aeruginosa has been held responsible for serious infections especially in hospitalized and immunocompromised patients. Three subtypes of type I CRISPR system (I-C, I-E, & I-F1) have been detected in P. aeruginosa genomes. In this work, P. aeruginosa isolates were collected from different clinical sources, and the three CRISPR/Cas subtypes (I-C, I-E, & I-F1) were detected via singleplex and multiplex PCR techniques using novel universal primers that were designed specifically in this study. CRISPR subtypes I-C, I-E, and I-F1 were detected in 10, 9, and 13 isolates, respectively. Furthermore, antimicrobial susceptibility of CRISPR/Cas-positive and negative isolates to different antibiotics and the capacity of biofilm formation were detected using disc diffusion method and tissue culture plate method, respectively. There was a significant correlation between the presence/absence of CRISPR/Cas system and both antimicrobial susceptibility to some antibiotics and biofilm-forming capacity among P. aeruginosa clinical isolates. KEY POINTS: • A novel multiplex-PCR for detection of CRISPR/Cas-positive strains of P. aeruginosa. • Understand the correlation between CRISPR/Cas systems and other characters of P. aeruginosa. • Correlation between antimicrobial susceptibility and CRISPR systems in P. aeruginosa., (© 2022. The Author(s).)

Escherichia coli is a multifaceted microbe since some are commensals, normally inhabiting the gut of both humans and animals while others are pathogenic responsible for a wide range of intestinal and extra-intestinal infections. It is one of the leading causes of septicemia, neonatal meningitis, urinary tract infections (UTIs), cystitis, pyelonephritis, and traveler's diarrhea. The present study aims to survey the distribution and unravel the association of phylotypes, virulence determinants, and antimicrobial resistance of E. coli isolated from different clinical sources in Mansoura hospitals, Egypt. One hundred and fifty E. coli isolates were collected from different clinical sources. Antimicrobial resistance profile, virulence determinants, and virulence encoding genes were detected. Moreover, phylogenetic and molecular typing using ERIC-PCR analysis was performed. Our results have revealed that phylogroup B2 (26.67%) with the greatest content in virulence traits was the most prevalent phylogenetic group. Different virulence profiles and varying incidence of virulence determinants were detected among tested isolates. High rates of resistance to different categories of antimicrobial agents, dramatic increase of MDR (92.67%), and emergence of XDR (4%) were detected. ERIC-PCR analysis revealed great diversity among tested isolates. There was no clustering of isolates according to resistance, virulence patterns, or phylotypes. Our research has demonstrated significant phylogenetic diversity of E. coli isolated from different clinical sources in Mansoura hospitals, Dakahlia governorate, Egypt. E. coli isolates are equipped with various virulence factors which contribute to their pathogenesis in human. The elevated rates of antimicrobial resistance and emergence of MDR and XDR mirror the trend detected globally in recent years. KEY POINTS: • Clinical E. coli isolates exhibited substantial molecular and phylogenetic diversity. • Elevated rates of antimicrobial resistance and emergence of XDR in pathogenic E. coli. • B2 Phylogroup with the highest VS was the most prevalent among pathogenic E. coli., (© 2022. The Author(s).)

Pseudomonas aeruginosa is a serious pathogen particularly in immunocompromised patients. In this work, 103 clinical isolates of P. aeruginosa were collected and classified into weak, moderate, and strong biofilm producers according to their biofilm forming abilities via tissue culture plate method. The antimicrobial resistance and the presence of different virulence genes were investigated via disc diffusion method and polymerase chain reaction respectively. Moreover, ERIC-PCR typing was performed to investigate the genetic diversity among the clinical isolates. No significant correlation was observed between biofilm formation and resistance to each antimicrobial agent. Similar observation was detected concerning the multidrug resistance and biofilm formation. Regarding virulence genes, algD gene was harbored by all isolates (100%). Only pelA and phzM were significantly prevalent in strong biofilm producers. Additionally, the mean virulence score was higher in strong biofilm producers (9.33) than moderate (8.62) and weak (7) biofilm producers. Moreover, there was a significant correlation between the overall virulence score of the isolates and its ability to form biofilm. ERIC-PCR genotyping revealed the presence of 99 different ERIC patterns based on 70% similarity, and the different ERIC patterns were categorized into 8 clusters. 100% similarity indicates the possibility of cross-colonization in P. aeruginosa infections., (Copyright © 2021. Published by Elsevier Ltd.)

Over the past decades, Escherichia coli (E. coli) have acquired extensive resistance to antibiotics; especially β- lactams. This study aimed to investigate the frequency of Extended-spectrum β-lactamase (ESBL) and carbapenemase producers among E. coli isolates and their correlation with serotypes, phylogenetic background, and pathogenicity associated islands. A total of 105 E. coli strains were isolated and subjected to antimicrobial susceptibility testing against β-lactam antibiotics. All isolates showed a high resistance profile. Resistant isolates were tested for ESBL and carbapenemase production. Fifty-three and 18 isolates were positive for ESBL and carbapenemase producers, respectively. ESBL and carbapenemase genes were detected by PCR. TEM gene was the most prevalent gene among all isolates followed by SHV and CTX-M15. In carbapenemase-producers, OXA-48 and IMP were the predominant genes. Enteropathogenic E. coli (EPEC) and Enterohemorrhagic E. coli (EHEC) were the major producers of ESBL and carbapenemase, respectively as indicated by serodiagnosis. They were further assessed for the presence of pathogenicity islands (PAIs) and phylogenetic background. The most predominant DEC PAI and ExPEC PAI were HPI and IICFT073. Most clinically ESBL-producers were group D and B2 while environmentally ones were group B1 and A. On contrary, clinically carbapenemase-producers belonged to group C and D. In conclusion, our study confirms the importance of phylogenetic group D, B2, and C origin for antibiotic resistance in E. coli. Ultimately, our findings support the fact that environmental isolates contribute to the local spread of E. coli pathogenicity in Egypt and these isolates maybe serve as reservoirs for transmission of resistance., Competing Interests: The authors have declared that no competing interests exist.

Pseudomonas aeruginosa is an opportunistic pathogen that can form biofilms, which confer resistance to immune clearance and antibacterial treatment. Therefore, effective strategies to prevent biofilm formation are warranted. Here, 103 P . aeruginosa clinical isolates were quantitatively screened for biofilm formation ability via the tissue culture plate method. The effects of lysozyme (hydrolytic enzyme) and proteinase K (protease) on biofilm formation were evaluated at different concentrations. Lysozyme (30  μ g/mL), but not proteinase K, significantly inhibited biofilm formation (19% inhibition). Treatment of 24-hour-old biofilms of P . aeruginosa isolates with 50 times the minimum inhibitory concentrations (MICs) of ceftazidime and cefepime significantly decreased the biofilm mass by 32.8% and 44%, respectively. Moreover, the exposure of 24-hour-old biofilms of P . aeruginosa isolates to lysozyme (30  μ g/mL) and 50 times MICs of ceftazidime or cefepime resulted in a significant reduction in biofilm mass as compared with the exposure to lysozyme or either antibacterial agent alone. The best antibiofilm effect (49.3%) was observed with the combination of lysozyme (30  μ g/mL) and 50 times MIC of cefepime. The promising antibiofilm activity observed after treatment with 50 times MIC of ceftazidime or cefepime alone or in combination with lysozyme (30  μ g/mL) is indicative of a novel strategy to eradicate pseudomonal biofilms in intravascular devices and contact lenses., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Mohamed Eladawy et al.)

Introduction: Emergence of carbapenem resistance in Pseudomonas aeruginosa increases the therapeutic dilemma. In this study, we investigated various mechanisms involved in the resistance of P. aeruginosa clinical isolates to carbapenems., Methodology: P. aeruginosa isolates were isolated from different clinical samples. The antimicrobial susceptibility was evaluated by disc diffusion method. Carbapenemases were detected among carbapenem resistant isolates. Expression level of mexB and oprD was determined by real-time PCR. Molecular relatedness among isolates was detected based on pulse-field gel electrophoresis (PFGE)., Results: Ninety P. aeruginosa isolates were purified from clinical specimens. High levels of resistance to imipenem and meropenem were detected in 16 isolates. PCR analysis of carbapenemases indicated the prevalence of Verona integron-encoded metallo-beta-lactamase (VIM); four isolates produced only VIM enzymes (VIM-1 or VIM-2), while the remaining twelve co-produced both VIM-1 or VIM-2 and NDM enzymes. Additionally, real-time PCR analysis elucidated high expression levels of mexB in seven of the carbapenem resistant isolates and low expression of oprD in seven isolates. The identified carbapenem-resistant isolates were clustered into eleven PFGE profiles where clusters E1 and E2 involved isolates exhibiting multiple carbapenemase genes (blaNDM-1, blaVIM-1 and blaVIM-2)., Conclusion: Various mechanisms underlying carbapenem resistance have been detected in our P. aeruginosa cohort of isolates. Emergence of P. aeruginosa as a reservoir of multiple carbapenemases is increasing over time limiting the treatment options to this serious infection. This increases the urgency for infection control practices to reduce the incidence of this infection., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2018 Mona Shaaban, Ahmed Al-Qahtani, Mohammed Al-Ahdal, Rasha Barwa.)

Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is a versatile pathogen capable of causing multitude of human diseases. It is one of the most important nosocomial pathogen that implicated in community and healthcare associated infections. Therefore, this study aims to characterize different SCCmec elements found in MRSA isolates. Moreover, molecular typing was performed to investigate the genetic relatedness among MRSA isolates., Methods: Phenotypic identification of MRSA was done by disc diffusion method. The MRSA isolates were typed based on the SCCmec, coa and agr genes. Phenotypic characterization included the detection of biofilm, lipase, protease, lecithinase, staphylokinase and hemagglutination. Also, hla, hlb, hlg, hld, tsst-1, psm-mec and mecI genes were detected genotypically. The correlation between the molecular types identified and the profile of virulence factors, clinical and geographical sources was determined for all isolates., Results: Eighty five isolates were identified as MRSA. Eight types of SCCmec elements were detected among these isolates. Type V was the most observed type (56.47%). Regarding the correlation between SCCmec types and virulence factors, type V SCCmec exhibited a significant association with biofilm (p < 0.0001), staphylokinase (p = 0.0495) and tsst-1 (p = 0.0498). Molecular typing of coa gave an insight to the presence of specific types in specific hospital wards. Based on agr typing, agr I was the highest prevalent type in MRSA isolates (54.11%)., Conclusion: There is an increase of MRSA infections particularly the community acquired with high variability in the distribution of virulence factors among different SCCmec types. The association between type III and V SCCmec with certain hospitals may be an evidence of nosocomial infection among these hospitals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Emergence of carbapenems-resistant K. pneumoniae represents a serious challenge for antimicrobial therapy., Objective: The aim of this research is to determine different mechanisms mediating the emergence of K. pneumoniae isolates with high-level carbapenem resistance., Method: A total of 80 K. pneumoniae isolates were purified from sputum and urine specimens. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by broth microdilution method. Carbapenemases were detected by Modified Hodge test and PCR. Additionally, the copy numbers of the identified genes ( bla VIM-1 , bla NDM-1 and bla OXA-48 ) were quantified by RT-PCR. The outer membrane proteins OmpK35 and OmpK36 of the resistant isolates were analyzed., Results: Eight isolates were resistant to carbapenems; six of these isolates possessed elevated MICs to imipenem and meropenem (≥16 µg/ml). Carbapenem resistant isolates harbored bla NDM-1 (n=5), bla VIM-1 (n=4) and bla OXA-48 (n=1) with some isolates had multiple carbapenemases genes. Six isolates with high MICs to imipenem contained multi-copies of the carbapenemases genes along with the lack of OmpK35. Isolates with intermediate resistance to carbapenems (MIC; 4-8 µg/ml) did not exhibit multiple carbapenemases but lacked the OmpK35. Random amplified polymorphic DNA exhibited three different patterns and indicated that five isolates encoded the same pattern P1., Conclusion: This study elucidated that multiple carbapenemases genes, high copy number of carbapenemases and loss of the porin OmpK35 could collectively contribute to the emergence of K. pneumoniae isolates with high resistance to carbapenems. Hence, more restrictions should be applied on the use of carbapenems to reduce the emergence of the resistant clones.

The spread of multidrug-resistant Pseudomonas aeruginosa isolates constitutes a serious clinical challenge. Bacterial efflux machinery is a crucial mechanism of resistance among P. aeruginosa. Efflux inhibitors such as phenylalanine arginyl β-naphthylamide (PAβN) promote the bacterial susceptibility to antimicrobial agents. The pathogenesis of P. aeruginosa is coordinated via quorum sensing (QS). This study aims to find out the impact of efflux pump inhibitor, PAβN, on QS and virulence attributes in clinical isolates of P. aeruginosa. P. aeruginosa isolates were purified from urine and wound samples, and the antimicrobial susceptibility was carried out by disc diffusion method. The multidrug-resistant and the virulent isolates U16, U21, W19 and W23 were selected. PAβN enhanced their susceptibility to most antimicrobial agents. PAβN reduced QS signalling molecules N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone without affecting bacterial viability. Moreover, PAβN eliminated their virulence factors such as elastase, protease, pyocyanin and bacterial motility. At the transcription level, PAβN significantly (P<0.01) diminished the relative expression of QS cascade (lasI, lasR, rhlI, rhlR, pqsA and pqsR) and QS regulated-type II secretory genes lasB (elastase) and toxA (exotoxin A) compared to the control untreated isolates U16 and U21. In addition, PAβN eliminated the relative expression of pelA (exopolysaccharides) in U16 and U21 isolates. Hence, P. aeruginosa-tested isolates became hypo-virulent upon using PAβN. PAβN significantly blocked the QS circuit and inhibited the virulence factors expressed by clinical isolates of P. aeruginosa. PAβN could be a prime substrate for development of QS inhibitors and prevention of P. aeruginosa pathogenicity.